1. Field of the Invention
The present invention relates to the production of the alkyl sulfate of sulfated dextrin, the production of the alkyl sulfate of sulfated dextran, and to the use of these compounds to provide antiviral activity, particularly in the treatment and prevention of sexually-transmitted diseases.
2. Description of Related Art
Compounds exhibiting activity against viruses may function by a number of mechanisms: they may kill or disable the disease pathogens, they may inhibit the entry of the pathogen into cells, or they may prevent replication of the pathogen once it has entered a cell. All of these mechanisms are being studied to prevent and treat viral infection, including those resulting in diseases that can be sexually transmitted, such as Acquired Immunodeficiency Disease Syndrome (AIDS).
The generally accepted theory is that AIDS is caused by the Human Immunodeficiency Virus (HIV). There are two different versions of HIV: HIV-1 and HIV-2. These viruses are believed, on the basis of their genetic sequences, to have evolved from the Simian Immunodeficiency Virus (SIV), with HIV-2 being much more similar to SIV. Several years after the initial HIV infection, the immune system is weakened to the point where opportunistic infections occur, resulting in the syndrome of AIDS.
Research has revealed a great deal of valuable medical, scientific, and public health information about HIV and AIDS. HIV molecules whose structures are known include reverse transcriptase (RT), proteases of HIV-1 and HIV-2, the catalytic domain of HIV integrase (INT), the HIV matrix protein, the HIV capsid protein and several fragments of CD4. HIV macromolecules whose structures are being investigated include the surface glycoproteins (gp160, gp120, gp41), and the regulatory proteins (tat, rev, vpr, tar).
The ways in which HIV can be transmitted have been clearly identified. HIV is spread by sexual contact with an infected person, by sharing needles and/or syringes (primarily for drug injection) with someone who is infected, or through transfusions of infected blood or blood clotting factors. Babies born to HIV-infected women may become infected before or during birth or through breast-feeding after birth. In the health care setting, workers have been infected with HIV after being stuck with needles containing HIV-infected blood or, less frequently, after infected blood gets into a worker""s open cut or a mucous membrane (for example, the eyes or inside of the nose). HIV is found in varying concentrations or amounts in blood, semen, vaginal fluid, breast milk, saliva, and tears.
In recent years, medical science has made great progress in the ability to successfully treat the opportunistic infections associated with HIV infection. Wider use of medications for preventing tuberculosis, Pneumocystis carinii pneumonia (PCP), toxoplasmosis, and Mycobacterium avium complex (MAC), for example, has helped reduce the number of people with HIV who develop serious illness and die from AIDS.
Also, several classes of compounds have been federally approved to treat HIV infection. These include nucleoside RT inhibitors (AZT, ddI, ddC, d4T and 3TC), non-nucleoside RT inhibitors (alpha-APA, TSAO, costatolide, TIBO, UC10), protease inhibitors (indinavir, saquinavir, KNI 272), attachment inhibitors (sulfate polysaccharides, sulfonated dyes) and neutralizing antibodies. Combinational therapy with these drugs seems to produce the best results, reducing the level of HIV particles circulating in the blood (viral load) to very low levels in many individuals.
Though treatment results using these drugs have been encouraging, the virus is not eliminated, these drugs do not work for all people, there are adverse interactions with other medications, toxicity to the drugs is problematic, dosing protocols are complex, resistance to treatment develops, and expense is extremely high. Furthermore, long-term effectiveness and safety are completely unknown. Clearly, there remains a need for new therapies.
Attempts to develop a vaccine have not been successful to this point.
Testing facilities perform in vitro analyses to identify compounds with antiviral activity. Therapeutic indices of active compounds are evaluated using several viral strains. Many viruses are routinely available for the testing of compounds for antiviral activity in viruses other than HIV, including the herpesviruses HSV-1, HSV-2, HCMV, VZV and EBV; the respiratory viruses Flu A, Flu B, RSV, Paraflu 3 and Ad5; Measles and Hepatitis B virus. Anti-HIV assays are routinely performed in established cell culture lines. Recently fresh human peripheral blood lymphocytes (PBMCs) have been introduced as test media.
Assays measure the ability of compounds to directly inactivate the HIV virus and inhibit HIV-induced cell killing through numerous enzyme-inhibiting mechanisms (Reverse Transcriptase, RNaseH, Integrase, Protease, Tat, Rev and Nef), by preventing attachment and internalization (inhibit gp120-CD4 interaction) or by inhibiting regulatory protein expression, or by inhibiting maturation and budding, or by preventing Syncytical formation. Toxicity of the test compounds to host cells is also measured. It is generally accepted that if the test compound is highly toxic to cells then it will have little value despite anti-HIV activity.
Infectious virus levels are measured by viral titers, quantitation of p24 (a viral protein found to be proportional to viral concentration) or measurement of the activity of the viral enzymes.
Several parameters are routinely varied to more completely understand the potential of a particular drug. The concentration of a drug is varied to calculate the ED50 (Effective Dose at 50% inhibition), LD50 (Lethal Dose at 50% cell death), and T150 (Therapeutic Index, which is the Effective Dose divided by the Lethal Dose).
The concentration of the initial viral load is varied in the cell system used for testing to help determine drug potency. The time of drug addition to the cell system, either pre- or post-infection, is varied to identify strengths and weaknesses in the drug mechanism of action. Another test is to add the drug to the cell system and then wash it away before infection. This gives insight into cell-drug mechanisms of action. Topical assays test drugs which may be of use as preventive barriers. Both viral killing and cellular toxicity are measured in these assays.
Active anti-HIV compounds will likely be used in combination with other anti-HIV agents, with agents that inhibit opportunistic agents, or with other therapies. Therefore, the compounds are tested with all known useful drugs to determine beneficial synergistic effects or possible harmful combinatorial toxicity.
Drugs that prove to be successful in in vitro testing are selected for animal testing. Several animal models have proven to be helpful including systems using the mouse, cat, and rhesus macaque. The test compound and virus can be administered by a variety of methods and routes in addition to the variables discussed above. Animal mucosal models of HIV transmission may be useful for the evaluation of possible therapeutic agents. Test compounds that may have limited effectiveness in fully developed HIV may be effective at the time of initial infection. Models are useful in exploring this possibility. Animal models traditionally have been used for the pre-clinical evaluation of lead compounds to determine mechanism of action, distribution, toxicity, and efficacy.
Antiviral compounds are also being investigated for use as microbicides. A xe2x80x9cmicrobicidexe2x80x9d is any substance that can substantially reduce transmission of sexually transmitted infections (STIs) when applied either in the vagina or rectum. Target viruses include herpes viruses such as cytomegalovirus and herpes simplex, hepatitis agents, and the papilloma virus. Proposed forms for microbicides include gels, creams, suppositories, films, and sponges or vaginal rings that slowly releases the active ingredient over time. Microbicides are not currently available commercially, but a number of compounds, including nonoxynol-9, cellulose sulfate, carrageenan, cyanovirin, the sulfated polysaccharide PRO2000 and dextrin sulfate, are currently being evaluated. Dextrin sulfate has been found to have a high level of toxicity. Nonoxynol-9 has been found to cause inflammation of mucosa that may actually enhance the chance of infection. There remains a need for a proven effective nonirritant antiviral compound of low toxicity for use as a microbicide.
Polysulfonated polysaccharides (PSP) have been previously proposed to be used to treat HIV infection. The most studied include curdlan sulfate (CDS), dextrin sulfate, dextran sulfate (DS), and heparin sulfate. Many of these, including dextran sulfate, curdlan sulfate and dextrin 2-sulfate, have been studied in human trials. Many other naturally occurring isolated sulfates have been shown to inhibit the AIDS virus. Smaller non-polymeric sulfated sugar based compounds included pentosan sulfate and glucosamine sulfate.
Though the results indicate that sulfates are a viable lead for the development of an anti-HIV drug, several problems remain. Firstly, the large anionic structures of the PSPs show very poor absorption or no absorption from oral administration. Secondly, when PSP""s are given intravenously the toxic effects of seriously decreasing the amount of platelets and decreasing the ability of blood to clot become limiting factors. Oral administration is also related to serious gastrointestinal toxic effects including the possible development of cancers demonstrated in rodents. Furthermore, there is no protection of the compounds from sulfatase enzymes which rapidly degrade these compounds and shorten the half-life.
The use of dextrin-2 sulfate as an anti-HIV compound versus generically sulfated dextrin (that is sulfates at any or all of the 2, 3 or 6 positions of the glucose units) has also been proposed The use of dextrin-2 sulfate is an attempt to decrease toxicity while maintaining anti-HIV activity. Recent attempts to administer dextrin-2 sulfate by intra-peritoneal administration (that is, infusion into the body cavity by a catheter passing through the abdominal wall as done in peritoneal dialysis) shows some promise in decreasing HIV infection while decreasing intravenous-type side effects. However, the intra-peritoneal method introduces extremely little if any drug to the systemic circulation and relies upon the lymphatic circulation to expose circulating HIV infected white blood cells to the drug as they pass through the peritoneal cavity. Evaluation of dextrin 2-sulfate shows that the anti-platelet effect and anti-coagulant effect persists and there is no attempt at chemical inhibition of the hydrolysis of the drug by hydrolyzing enzymes. Consequently, dextrin 2-sulfate has not been shown to provide significant advantages over dextrin sulfate.
Accordingly, there remains a need to identify and synthesize a compound with minimized toxicity, providing antiviral activity including, but not limited to, microbicidal activity. There remains a need for a pharmaceutical composition incorporating this compound, and for methods of treatment, inhibition of viral transmission, and elimination of virus in blood, blood products, organs and whole body preparations incorporating this compound.
The present invention is directed to a new class of compounds, methods for their synthesis, and to the use of these compounds in providing antiviral activity. This class of compounds is produced by alkylsulfation (alkylsulfonation) and sulfation (sulfonation) of dextrin or dextran. The reaction used introduces aliphatic alkyl groups and sulfur groups onto a carbohydrate or polysaccharide. This reaction randomly replaces the reactive hydrogen atoms with a methylsulfate group or a sulfate group and allows for a combinatorial production of sulfate and methylsulfate substitution of dextrin or dextran. The variables of this reaction that can be controlled include the choice of dextrin or dextran as a reactant, the polymeric size of the starting material, the degree of total methylsulfation and sulfation, the degree of methylsulfation and sulfation per saccharide, and position of methylsulfation and sulfation (sulfonation) and the character of the counter ion. Control of these variables, along with the polymeric size of the starting material and degree of hydrolysis during the reaction or work-up, produces a wide range of polymeric compounds. These new compounds are distinctly different from other compounds introduced for anti-HIV therapy. These compounds have a unique synthesis, unique chemical properties and a unique pattern of activity against HIV. Use of these compounds overcomes the absorption obstacles, toxicity obstacles, and efficacy obstacles presented by prior art compounds while retaining the anti-HIV properties of sulfated saccharides. Use of the compounds of the present invention, incorporating alkyl sulfonate groups, embodies the realization that these obstacles are related to the linear sulfated structures and the non-attenuated high degree of anionicity characteristic of these sulfated compounds, and the lack of the presence of an inhibitor to enzymatic sulfate hydrolysis.
The invention introduces four important changes. Firstly, the crucial structural element required for anti-HIV activity is recognized to be the cluster of sulfate groups presented on the branch point structures. Secondly, the structural element of toxic side effects is recognized as the sulfate groups on the linear portions. Elimination of linear portions and amplification of branch point sulfated structures decreases toxic side effects and increases therapeutic effects. Thirdly, introduction of the methylsulfate group in synergy with the sulfate group increases efficacy by several possible mechanisms, including the providing of an inhibitor to sulfate hydrolyzing enzymes, the attenuation of the large negative charge and the proposed increase in oral, systemic and cellular absorption and efficacy. Finally, the number of sulfated structures or combinations of structures provides variable sites for binding and enzyme inhibition.
The antiviral activity of the compounds of the present invention is explained here in relation to, but is not limited to, the Human Immunodeficiency Virus (HIV). The generally accepted theory is that Acquired Immunodeficiency Disease Syndrome (AIDS) is caused by the Human Immunodeficiency Virus (HIV) and that the prevention of the reproduction of HIV will prevent AIDS. The reproduction of the virus relies on the function of the reverse transcriptase enzyme (RT). RT function requires the binding protein Trans Activating Transcriptor (TAT). The present invention prevents the reproduction of HIV by binding with the TAT protein and preventing the proper function of RT.
The comparatively low toxicity and comparative absence of detrimental effects on body tissue allow the use of the compounds of the present invention in a number of applications calling for compounds exhibiting antiviral activity. The compounds may be used directly, alone or in combination with other therapy, as an anti-viral or anti-HIV drug. The compounds of the present invention may also be used in preventative treatments for HIV or other viruses. Routes of administration for these uses include oral and topical administration, and sub-cutaneous, muscular, intraperitoneal or intravenous injection. The compounds of the present invention may be used in bound and unbound form to eliminate HIV or other viruses from blood products during dialysis of organ or whole body preparations. They may also be used alone or in combination in cell culture systems or organ preservation systems to destroy or prevent HIV or other viral growth.